In many transplantation-type situations, there is concern for differences between the allotype, especially the HLA tissue type, of a cell source and the cell recipient. Antibodies against HLA can be induced by multiple blood transfusions, pregnancy, or during a prior transplant rejection. Although these antibodies may be low titer, and difficult to detect, their presence in the blood of a potential recipient is indicative that a new transplant with matching HLA could be rejected. The determination of the presence and specificity of antibodies against foreign HLA is therefore clinically important for monitoring transplant candidates and patients. Detection assays have tested for reactivity against a panel of lymphocytes, as an initial broad screen (panel reactive antibodies, PRA testing), or may be specific for a single donor (donor specific crossmatch).
The detection of antibodies specific for HLA is useful in situations such as transplantation, and platelet transfusion. Patients awaiting transplantation of kidney, liver or other organs usually have their blood tested monthly, or at least quarterly for antibodies against HLA. Because the patients occasionally need transfusions of blood or blood products, have had a pregnancy, or have received a prior organ transplant, there is opportunity for a patient to be immunized to the HLA of other people.
In a known method, a sample is collected from a patient and tested against individual white cell samples of 40-80 different people to determine 1) if antibody has been made against foreign HLA, and 2) which antigens are detected by the patient's antibody. There are hundreds of possibilities since each person's white cells carry 8 HLA Class I tissue antigens and 2 Class II tissue antigens.
A standard technique for HLA typing and detection of anti-HLA antibodies is microlymphotoxicity, where serum containing antibodies is incubated with HLA antigen-expressing lymphocytes, then with complement. If antibodies recognize a specific HLA and attach to lymphocyte cells, the cells activate the circulatory complement components. Once activated, complement components are lytic and cause the cells to become porous and die. In some cases anti-human immunoglobulin is added to augment cell killing. When the porous cells die, they can no longer keep a dye from entering them. The technologist who looks at the cells in a microscope sees dark, swollen, dye laden cells if they are dead or bright refractile cells if they are living. The level of cytotoxicity is estimated by discriminating between dead and viable cells using various dyes. The technologist records a grade of 1, 2, 4, 6, or 8 to indicate how many of the cells in that test are dead. A grade of 8 is positive and means that 80-100% of the cells have been killed. Each grade below 8 is assigned based on the percent of dead cells observed. The number assignment is an estimate rather than an exact count.
When the cells of 40-80 different donors have been examined and the results recorded, the technologist attempts to determine what antigen(s) the dead cells have in common. The tissue types of the dead cells are compared. This method has numerous disadvantages: it is labor intensive, time consuming, requires isolation of cells, requires viable cells, is nonspecific for HLA, and requires a subjective evaluation.
Flow cytometry may also be used to detect antibody-HLA activity but has many of the same disadvantages as cytotoxicity and requires expensive instrumentation. The analysis is extremely complicated and requires several years of experience to be able to interpret successfully. One difficulty in interpretation occurs when patients have multiple HLA antibodies at one time. In those instances, all cells in the test receive a grade of 8 which means that no antibodies can be identified since all tests are equally positive. In these cases, the antibody is called "multispecific".
Therefore it is important to provide alternative techniques which can be performed simply, can be automated, do not share the shortcomings described, provide a readily discernible result which is significant for the prognosis of transplant acceptance, and are comparable to data from existing tests.
Methods of HLA typing have been previously described. U.S. Pat. No. 5,223,397 describes a method of determining HLA cross match with a soluble form of HLA molecules found in biological samples. U.S. Pat. No. 5,292,641 describes the detection on a solid support of HLA or HLA specific antibodies using antigen shed from cell surfaces, called soluble antigen.